Pluripotent stem cells such as embryonic stem cells (ESCs) and embryonic germ cells (EGCs) have the unique abilities to both self renew indefinitely as well as being able to give rise to most, if not all cell types present in the human body. Given these properties, only a few factors that control their growth as undifferentiated cells have been identified. Indeed, many attempts have been reported to elucidate the mechanisms involved using transcriptome comparisons among various ESC lines and other cell types. These studies provide the basis for this proposal. However, this project proposes a novel model to identify potential factors in pluripotency by studying the genes involved in the conversion of primordial germ cells (PGCs) into EGCs. This model is unique in that unlike ESCs, the specific cell type of origin of EGCs is known and that these cells, PGCs can be isolated for further study. Specifically, in Aim 1 genomic comparisons will be performed among human PGCs isolated from cell culture at sequential time points in their conversion to pluripotent EGCs while Aim 2 will study genomic alterations in PGCs versus undifferentiated EGCs and ESCs (WA01) and those undergoing differentiation. Data from PGCs will also be compared to those made between feeder sublines which are distinguished only by their functional ability to derive EGCs. This comparison will provide additional information for identifying genes of interest by focusing on complementary systems between PGCs and feeder cells to help prioritize candidate genes. Using this model, candidate genes will be selected using the following prioritization (1) transmembrane proteins that complement ligands identified from mouse embryonic feeder layers which support EGC derivation;(2) genes associated with pluripotency in other species;(3) those containing sequence elements that may be responsive to known pluripotent factors such as Oct4, Nanog, or Sox2;and (4) finally, pathways shown to be relevant in pluripotent or multipotent cells. Expression of selected candidates will be validated using standard quantitative nucleic acid and protein analyses. In Aim 3, functional roles will then be performed by conditional knock-in or knock-down gene approaches in addition to cell culture manipulations using growth factors and their inhibitors. Importantly, this proposal demonstrates the feasibility of obtaining early germ cells of human origin in sufficient quantities and to obtain biologically relevant data. Data which may also suggest a common origin for EGCs and ESCs. Furthermore, the PGC-EGC conversion model provides for efficient in vitro functional assays to test candidate factors which will optimize the culturing of EGCs and provide insight into genes involved in the pluripotential conversion of PGCs to EGCs that will help fill gaps in our understanding of pluripotent stem cell derivation and maintenance. PUBLIC HEALTH RELEVANCE: With their unique abilities of unlimited self-renewal and to give rise to most, if not all cell types present in the human body, pluripotent stem cells have enormous promise for the treatment of human disease. However, despite demonstrations of their potential use in cell-based therapies, little is known regarding the regulation of their growth as undifferentiated cells -an issue critical for maximizing embryonic and adult stem cell utilization. For this purpose, the following study will use an exploratory approach involving genomic comparisons among pluripotent stem cells and an unipotent progenitor population to help identify mechanisms altered in the their progression toward the pluripotent state.